1. Field of the Invention
This invention relates to devices and methods for performing assays to determine the presence or quantity of a specific analyte of interest in a fluid sample. Devices of this invention assay a measured amount of sample employing at least two separate and distinct flow paths which are initiated simultaneously with a single user activation step. These paths are timed for the sequential delivery of assay reagents to the reaction zone, followed by wash or substrate and wash reagents to that zone. These inventive devices and methods may be used for qualitative, semi-quantitative and quantitative determinations of one or multiple analytes in a single test format. They may be practiced with ELISA, sol particle and other assay formats, and are particularly suitable for simultaneous multiple analyte assays. These inventive devices and methods provide for the controlled, self delivery of reagents with no timed steps, and minimal user intervention, in most instances a single activation step.
2. Background
Many prior art assay devices and systems require the user to measure or control the amount of sample added to the device, for example, by dilution. Many prior art assays and systems also require the user to perform a timed sequence of steps and/or to make multiple physical interventions to the device in order to perform the assay.
Buechler et al., U.S. Pat. Nos. 5,458,852 and 5,885,527 (1995 and 1999, respectively), disclose diagnostic devices which do not use porous membranes. In assay methods using such devices, fluid flow is unidirectional and reaction and detection occur in distinct zones. Excess sample/conjugate mixture is used to wash the detection zone. The use of a separate, non-sample wash is not taught or suggested, limiting the versatility of assays of this invention. Likewise, Buechler does not teach or suggest the creation of a second flow path. The time gate in Buechler functions as a delay mechanism in a fluid path, not to redirect fluid flow or to permit different fluids to flow sequentially through a reaction zone. The methods and devices of Buechler et al. do not allow one to use multiple reagents, different wash and substrate reagents and cannot be used in an enzyme amplified assay.
Vonk, U.S. Pat. No., 5,185,127 discloses a filter stack comprising a hydrophilic membrane (containing a binder for an analyte) above a hydrophobic membrane above an absorbent material. The membrane is impermeable to the sample but permeable to a wetting agent (e.g. acetone, surfactants, detergents, or alcohols, most preferably methanol) mixed with the sample. Sample is added to the device and trapped above the hydrophobic member. Thereafter the wetting agent is added, which when mixed, allows the sample mixture to permeate into the absorbent material. Wash and substrate may be added. Other disadvantages of this device are that it (1) does not teach or suggest a defined fluid path for residual sample or wash, (2) does not permit controlled delivery of predetermined amounts of reaction components or wash, and (3) does not permit sequential delivery of such components.
Clark, U.S. Pat. Nos. 5,726,010, 5,726,013 and 5,750,333 (1998) describes assay methods and devices that use the formation of a solid phase bound tertiary complex to detect an analyte of interest in a fluid sample. A key feature of Clark is the use of a reversible flow in a chromatographic binding assay. An analyte-containing solution is applied to the device and then is transported by capillary flow, first in one direction and then in the opposite direction, along an elongated flow matrix. The flow matrix includes four different regions. Region one is where the analyte-containing solution is mixed with a labeled antibody. Region two, also called the detection zone, contains the second antibody, which is immobilized to a solid phase. Region three contains a site to apply a wash solution. Region four contains an absorbent reservoir located near region one and makes the flow go in the opposite direction. A means to detect the presence or quantity of an analyte is also included in the device. Clark does not automate steps. The user must measure the sample volume, apply it to the device, monitor and control timers, and physically activate the device. The mechanism for reversing flow does not allow for automated timing.
Assays and devices of the present invention overcome the shortcomings of the prior art and offer maximum results without user sample measurement or intervention beyond a single activation step. Assays of this invention may be particularly adaptable to situations where simultaneous detection of multiple analytes in a sample is desirable. Assays of this invention are in a unit dose format, stable, capable of room temperature storage, reliable, easy to manufacture and use, and available for a low cost per test. They have fully integrated packaging for both liquid and dried reagents. In addition, the claimed devices are self-timing for the delivery of reagents so there is minimal operator involvement.
Analytexe2x80x94The molecule to be detected. For example, an analyte as used herein, may be a ligand, a single compound or a plurality of compounds that share at least one epitopic site to a receptor or an antibody.
Capillarityxe2x80x94The movement of a liquid in contact with a solid that results due to adhesive and cohesive forces and surface tension. Capillarity can be affected by the solid surface, the liquid surface, or both.
Hydrophobic surfacexe2x80x94Any surface not effectively wetted by water or an aqueous sample.
Hydrophillic surfacexe2x80x94Any surface wetted by water or an aqueous sample.
Conjugate soluble binding reagentxe2x80x94Reagent(s) deposited and dried on a solid support, i.e., the sample delivery channel, and have a specific binding affinity for or chemical reactivity with the analyte of interest. Upon sample application, the conjugate soluble binding reagent becomes dissolved and can begin to flow. Conjugate soluble binding reagent can react with analyte, if present in the sample. Depending upon the assay format, one conjugate soluble binding reagent can be labeled with a detectable label and another with a binding reagent. These different assay formats will be described in more detail below.
Solid phase zonexe2x80x94A material or a surface at the intersection of at least two fluid flow paths.
Sample/conjugate mixturexe2x80x94The liquid mixture comprising the sample solution and solubilized conjugate binding reagents.
Immobilized capture reagentxe2x80x94A molecule that is bound to a solid support and has a specific binding affinity for or chemical reactivity with either the analyte of interest, or can be a receptor for one of the conjugate reagents e.g. avidin.
Sample delivery channelxe2x80x94The hydrophilic means where the conjugate soluble binding reagents are dried and the means through which the liquid sample flows.
Sockletxe2x80x94The hydrophilic mesh material that contains the solid phase when a particulate solid phase is used.
Solid phasexe2x80x94The hydrophilic material to which the immobilized capture reagent is bound.
Second fluid path materialxe2x80x94The hydrophobic material that provides the secondary flow path. Examples of second fluid path material are bibulous material, plastic, or any other hydrophobic polymers. As the surface active agent-containing wash solution flows through the second fluid path material, the surface tension is reduced and allows sample/conjugate mixture to flow through the second fluid path material, which is now rendered hydrophilic.
Wash reagentxe2x80x94A liquid reagent that serves to remove unbound material from the solid phase region. As used herein, the wash reagent contains a surface active agent, such as a surfactant, or any other component capable of allowing the wash to wet a hydrophobic surface. Some other examples of wash reagents are alcohol, e.g. methanol, or any other water miscible organic solvents.
Substrate reagentxe2x80x94A liquid reagent that serves to facilitate analyte detection by causing a detectable color reaction. A secondary function of substrate reagent is to remove unbound material from the solid phase region.
Wash/substrate reagentxe2x80x94In some embodiments of the present invention, the wash reagent may also contain a detection agent so that a single solution is delivered via said second flow path to the solid phase zone.
Fluidic bridgexe2x80x94The fluid flow connection that is established between the socklet, second fluid path material and the absorbent reservoir. The fluidic bridge enables liquid flowing in the second flow path to go into the absorbent reservoir.
Lance/wickxe2x80x94A component that is capable of piercing the seal of the liquid reagent containers, i.e., the wash and substrate reagent containers or the combined wash/substrate reagent container. The lance may also include a wick, which facilitates the flow of the liquid reagents out of their storage containers and into the second fluid path material.
The present invention is directed to devices and methods for performing an assay to determine the presence or quantity of a specific analyte of interest in a fluid sample. Devices according to this invention comprise a solid phase capable of capturing the analyte of interest after washing unbound material from the solid phase. In one aspect of the device two separate flow paths are established sequentially in the device with a single user activation step. The first flow path delivers the analyte of interest (if present in the sample) and conjugate soluble binding reagents to the solid phase. If analyte is present, an analyte:conjugate complex is formed and immobilized.
The volume of sample delivered by this first path is determined by the absorbent capacity of the solid phase, and not by the amount of sample added to the device, relieving the user from the necessity of measuring the sample. The sample/conjugate mixture is prevented from entering the second flow path because the capillarity and the surface energy of the second flow path prevent it from being wetted by this mixture.
The second flow path allows a wash reagent to remove unbound conjugate and sample from the solid phase to the absorbant, and optionally to deliver detection reagents. The flow of wash reagent along the second flow path is initiated by user activation of the device immediately after addition of sample to the first flow path. When wash reagent migrates to the solid phase, and flows through and around it, it removes unbound reagents and carries them to the absorbent. As a result, the incubation time of the sample and reagents in the solid phase is determined by the flow time of the wash reagent along the second flow path, relieving the user of the requirement to intervene at a specific time.
In one embodiment, the first flow path consists of a sample entry port, a filter element to remove particulates, an open capillary channel containing dried reagents, and a particulate solid phase. The second flow path intersects the first, and consists of a wash reservoir, an absorbent block, and a second fluid path material that extends from the reservoir, contacts the solid phase and finally contacts the absorbent. Sample is prevented from flowing into the second fluid path and absorbent because the surface energy of the second fluid path material is low relative to the sample surface tension, rendering it hydrophobic. The wash solution contains surfactant that allows it to wet the second fluid path material, and thus allow the sample-binding reagent mixture from the solid phase to also flow into the second fluid path material.
The invention may be adapted to many assay formats including, sandwich immunoassays, colloidal gold, or sol particle assays, heterogeneous generic capture assays and competitive assays.
In one embodiment, sandwich assays can be performed by immobilizing an analyte binding reagent on the solid phase, and drying a labeled analyte binding reagent in the first flow path. In a competitive assay embodiment, the first flow path would contain labeled analyte that is dissolved by the sample, and the analyte binding reagent is immobilized on the solid phase. In each of these embodiments, the assay can be further modified to run in a xe2x80x9cgeneric capturexe2x80x9d format, where the solid phase binding reagent is instead conjugated to a generic ligand such as biotin, and dried in the first flow path (either together or separately from the other assay reagents), and a generic ligand binding reagent (such as avidin) is immobilized on the solid phase.
Another aspect of the present invention includes a subassembly for the immunoassay device that is comprised of a plastic housing and a means for delivering fluid and/or wash solution, This subassembly comprises a structure formed from a hydrophobic polymer. The hydrophobic polymer has been selectively treated with a water insoluble surface active agent that has been applied as a solution in an organic solvent rendering portions of the surface hydrophilic. When the surface is contacted with an aqueous liquid, it flows only along the treated areas, creating a defined fluid flow path, thereby delivering sample/conjugate solutions to said solid phase.
Another aspect of the claimed device includes a solid phase subassembly referred to as a socklet. The socklet is comprised of a particulate solid phase material that is captured within a structure that is permeable to the sample and liquid reagents, but which prevents the particulate solid phase material from exiting the structure.